Biochemistry 2002, 41, 7895-7906
7895
Residues in the Subunit of the Nicotinic Acetylcholine Receptor Interact To Confer Selectivity of Waglerin-1 for the R- Subunit Interface Site† Brian E. Molles,‡,§ Igor Tsigelny,‡ Phuong D. Nguyen,‡ Sarah X. Gao,‡ Steven M. Sine,| and Palmer Taylor*,‡ Department of Pharmacology and Biomedical Sciences Graduate Program, UniVersity of California at San Diego, La Jolla, California 92093-0636, and Receptor Biology Laboratory, Department of Physiology and Biophysics, Mayo Foundation, Rochester, Minnesota 55905 ReceiVed February 25, 2002; ReVised Manuscript ReceiVed April 29, 2002
ABSTRACT: Waglerin-1 (Wtx-1) is a 22-amino acid peptide that competitively antagonizes muscle nicotinic acetylcholine receptors (nAChRs). Previous work demonstrated that Wtx-1 binds to mouse nAChRs with higher affinity than receptors from rats or humans, and distinguished residues in R and subunits that govern the species selectivity. These studies also showed that Wtx-1 binds selectively to the R- binding site with significantly higher affinity than to the R-δ binding site. Here we identify residues at equivalent positions in the , γ, and δ subunits that govern Wtx-1 selectivity for one of the two binding sites on the nAChR pentamer. Using a series of chimeric and point mutant subunits, we show that residues Gly-57, Asp-59, Tyr-111, Tyr-115, and Asp-173 of the subunit account predominantly for the 3700-fold higher affinity of the R- site relative to that of the R-γ site. Similarly, we find that residues Lys-34, Gly-57, Asp-59, and Asp-173 account predominantly for the high affinity of the R- site relative to that of the R-δ site. Analysis of combinations of point mutations reveals that Asp-173 in the subunit is required together with the remaining determinants in the subunit to achieve Wtx-1 selectivity. In particular, Lys-34 interacts with Asp-173 to confer high affinity, resulting in a ∆∆GINT of -2.3 kcal/mol in the subunit and a ∆∆GINT of -1.3 kcal/mol in the δ subunit. Asp-173 is part of a nonhomologous insertion not found in the acetylcholine binding protein structure. The key role of this insertion in Wtx-1 selectivity indicates that it is proximal to the ligand binding site. We use the binding and interaction energies for Wtx-1 to generate structural models of the R-, R-γ, and R-δ binding sites containing the nonhomologous insertion.
The nicotinic receptor contains five separate polypeptide subunits arranged with radial symmetry around a central channel (1, 2). The five subunits in the muscle receptor, two copies of R and one each of β, δ, and γ (embryonic subtype) or (adult subtype) (3), assemble in the following counterclockwise order: R-γ/-R-δ-β. The crystal structure of the acetylcholine binding protein (AChBP)1 from the freshwater snail Lymnaea stagnalis (4, 5) firmly establishes binding site location and subunit handedness in the nAChR by demonstrating that binding site residues contributed by the R subunit contribution are found on the “clockwise” face of the AChBP subunit, and the non-R subunit residues contributing to the binding site are found on the “counterclockwise” face of the AChBP subunit (6). The two binding sites for agonists, reversible competitive antagonists and the pseudo-irreversible R-bungarotoxin (R† This work was supported by Grants GM18360 to P.T., GM07752 to B.E.M., and NS31744 to S.M.S. * To whom correspondence should be addressed. ‡ Department of Pharmacology, University of California at San Diego. § Biomedical Sciences Graduate Program, University of California at San Diego. | Mayo Foundation. 1 Abbreviations: nAChR, nicotinic acetylcholine receptor; [125I]-RBgTx, [125I]-R-bungarotoxin; AChBP, acetylcholine binding protein; Wtx-1, waglerin-1.
BgTx), are formed at R-δ and R- (or R-γ) subunit interfaces of the muscle receptor. Mutagenesis and sitedirected labeling studies established that seven segments, far apart in the linear sequence, contribute to the ACh binding site: segments A-C in the R subunit and segments D-G in δ, , or γ subunits (1, 2). The AChBP structure confirmed that residues in each of these segments are present at the ACh binding site, establishing it as a template for the tertiary structure of the major extracellular domain. Each of the five subunits of the muscle nAChR contains between 445 and 497 amino acids. Each subunit has one to three N-linked glycosylation sites and four transmembrane spans, giving the pentamer a molecular mass of nearly 300 kDa. Its large size and amphipathic character make the nAChR refractory to crystallization and NMR spectroscopy aimed at determining its atomic structure. Isolated from the venom of Wagler’s pit viper, Tropidolaemus wagleri (7), the waglerin peptides are remarkably selective peptide antagonists. Site-selective antagonists are significant because although binding of two agonist molecules is required to activate the receptor, binding of a single agonist molecule blocks activation. The four closely related waglerins are the only characterized toxins from the Viperidae family of snakes that target nicotinic receptors; waglerin-1 (Wtx-1) has the sequence NH2-GGKPDLRPCH-
10.1021/bi025732d CCC: $22.00 © 2002 American Chemical Society Published on Web 05/29/2002
7896 Biochemistry, Vol. 41, No. 25, 2002 PPCHYIPRPKPR-COOH. A single intramolecular disulfide bond forms between the two Cys residues, as determined by NMR spectroscopy (8, 9). Wtx-1 binds to the adult mouse AChR with 2100-fold higher affinity for the R- over the R-δ binding site (10). In this regard, Wtx-1 joins Dtubocurarine (11), R-conotoxin MI (12, 13), and Naja mossambica mossambica R-neurotoxin (14) as highly siteselective prototype competitive antagonists against mammalian muscle receptors. Studies using these ligands demonstrated that the ligand binding sites are formed at interfaces between R and non-R subunits (11, 13-16). By quantification of contributions of individual residues to site selectivity for these ligands, residues on both the R and non-R subunits have been shown not only to be proximal to the bound ligand but also to govern the shape of the binding site and orientation of bound ligand. Here we use chimeric and point mutant subunits to identify residues in mouse , γ, and δ subunits that confer binding site selectivity for Wtx-1. Analysis of combinations of point mutations demonstrates that several of these determinants of ligand selectivity are energetically coupled, revealing cooperative participation in ligand recognition. Energetic coupling suggests the proximity of these residues to each other and to the ligand binding site, which enabled modeling of the binding sites using the crystal structure of AChBP (4, 5) as a template. EXPERIMENTAL PROCEDURES Synthesis and Purification of Waglerin-1. The crude peptides, synthesized by American Peptide Co. (San Jose, CA) or Synpep (Dublin, CA), were dissolved to a concentration of 0.8 mg/mL in 30 mM Tris-HCl (pH 8.2-8.5), sterile filtered, and left overnight at room temperature for formation of the single intramolecular disulfide bond in the Wtx-1 structure. After disulfide bond cyclization, 0.1% trifluoroacetic acid was added to minimize the formation of intermolecular disulfide bonds. Peptide solutions (2 mL) were loaded onto a 5 mL HPLC sample loop leading to a 10 mm × 250 mm semipreparative reverse-phase C18 column (Vydac), with 0.1% trifluoroacetic acid as solvent A and 0.085% trifluoroacetic acid and 70% acetonitrile as solvent B. Elution was achieved by increasing the level of solvent B from 20 to 25% over the course of 15 min. The cyclized peptide elutes 1-2 min earlier than uncyclized peptides or dimerized peptides formed by intermolecular disulfide bond formation (17). Fractions containing the purified peptide were pooled and lyophilized. Representative samples from different lots were checked for purity and correct mass by MALDI or ion-spray mass spectrometry. Mutagenesis of nAChR Subunits. Cloned cDNAs for mouse R (18), β (19), γ (20), δ (21), and subunits (22) were ligated into the mammalian expression vector pRBG4 at EcoRI sites for transient expression in HEK293 cells (11). Site-specific mutants of the wild-type mouse γ, δ, or subunit were made by one of two methods. Complementary synthetic oligonucleotides (Sigma/Genosys, The Woodlands, TX) containing the desired mutation were ligated into the cDNA at unique restriction sites flanking the mutated region. When convenient restriction sites were not available, the Stratagene double primer method was used. In this procedure, complementary oligonucleotides containing the desired mu-
Molles et al. tation served as primers in a PCR amplification reaction that included the wild-type plasmid and Pfu polymerase. Following 16-18 amplification cycles to make the entire plasmid, the reaction mixture was treated with DpnI restriction endonuclease to digest only the methylated, wild-type plasmid DNA, leaving behind the newly synthesized DNA. Mutations were subcloned into vectors not subjected to mutagenesis and verified initially by restriction digests and then by DNA sequencing. Large-scale plasmid preparations used DEAE columns (Gibco or Qiagen) or cesium chloride ultracentrifugation for purification. Transfections. The individual subunit cDNAs for the nAChR are contained on unique plasmids using the CMV promoter-based pRBG4 vector (23). HEK293 cells were transfected by the calcium phosphate method; the medium was changed 12-16 h later, and binding assays were performed 1-2 days after the medium change. Expression levels that allowed an estimation of Wtx-1 KD values ranged from 60 to 950 fmol per 10 cm plate of cells. Estimation of Waglerin-1 KD Values by Competition with the Initial Rate of [125I]-R-Bungarotoxin ([125I]-R-BgTx) Binding. Transfected cells were removed from the culture plates by gentle agitation with 5 mL of phosphate-buffered saline (pH 7.4) containing 5 mM EDTA. After incubation of the dissociated cells with waglerin-1 for 45 min to 1 h, 5 nM [125I]-R-BgTx (New England Nuclear) was added and allowed to incubate for 30 min such that 30-50% of the available binding sites become occupied (24). Waglerin dissociation constants were determined from the fractional reduction of the initial rate of [125I]-R-BgTx binding. The total number of sites was determined by incubating with 20 nM [125I]-R-BgTx for 1 h. The level of nonspecific binding was determined by incubating the cells with 10 mM carbachol before reacting with [125I]-R-BgTx. Transformed data were fit to either the one-site or two-site Hill equation using Prism 2.0 (Graphpad). Receptor Model Construction. The receptor was modeled structurally against the crystallographic coordinates of the acetylcholine binding protein (PDB entry 1i9b) using the program Homology and Insight II (Accelrys, 2000). The receptor structure was then energy minimized for 10 000 iterations of conjugated gradients using a distance-dependent dielectric constant with the program Discover (Accelrys, 2000). RESULTS Throughout this investigation, our working hypothesis is the protein scaffolds of the , γ, and δ subunits are, to a first approximation, superimposable; residues at homologous positions in the primary sequences occupy equivalent locations in three-dimensional coordinate space. Because each of these non-R subunits partners with an R subunit to form a binding site, they are the predominant sources of binding site selectivity for Wtx-1. Therefore, to identify determinants of Wtx-1 selectivity, our experiments seek to reconstitute binding affinity conferred by one non-R subunit by residue substitution into the template of another. In particular, we convert selectivity of the subunit into γ- and δ-like subunit, and convert the γ and δ subunit selectivity into an -like subunit. γ- Subunit Determinants for Waglerin. Because the γ and subunits show the highest level of identity among the
Waglerin-1 Binding to Nicotinic Receptors
Biochemistry, Vol. 41, No. 25, 2002 7897
FIGURE 1: Multiple alignment of the acetylcholine binding protein (AChBP) with the R, , δ, and γ subunits of the muscle nicotinic acetylcholine receptor. Highlighting in dark blue indicates identity and light blue similarity in at least three of the five sequences. Goldlettered residues are conserved in all five sequences. Numbers above the sequences are for the subunit, with the green numbers indicating the selectivity-determining residues. The regions of AChBP secondary structure (4) are indicated above the numbers, with R and 310 (labeled η) helices in red and β sheets in turquoise. The β8 sheet found in the AChBP, indicated with a hatched arrow, is not present in the model of the subunit shown in Figure 6. A neighboring R helix (yellow) is modeled in the insertion region between residues 158 and 167 that is not represented in the AChBP (4).
non-R subunits, are located between the two R subunits in the respective fetal or adult receptor, and confer very different affinities for Wtx-1, we initially studied a series of chimeras composed of portions of γ and subunits from the mouse nAChR. Each chimera was cotransfected with wild-type R, β, and δ subunits, and Wtx-1 affinity was assessed by competition against the initial rate of [125I]-R-BgTx binding (Figure 2 and Table 1). Throughout the text, chimeras are named starting with the subunit source of the N-terminal sequence, followed by the position of the chimeric junction, and ending with the subunit source of the C-terminal sequence. For example, the γ74 chimera contains residues 1-74 of the γ subunit spliced to residues 75-473 of the subunit. The γ74 chimera reduced affinity 82-fold compared to that of the wild-type subunit, indicating one or more determinants of selectivity are located within the first 74 residues. The γ103 chimera yielded essentially the same reduction in affinity as γ74, suggesting no additional determinants between positions 74 and 103. The γ117 chimera replicated the low affinity conferred by the γ subunit, giving a monophasic competition curve and a KD of 28 µM. These results suggest that residues conferring the affinity difference between γ and subunits are located within the first 117 residues of the subunit. However, the γ165 and γ171 chimeras further altered Wtx-1 affinity; γ165 increased affinity 3-fold compared to that of γ117, whereas γ177 decreased affinity to mimic the low-affinity characteristic of the wild-type γ subunit. The 65γ76 chimera gave an affinity identical to that of wild-
FIGURE 2: Waglerin-1 competition with the initial rate of [125I]RBgTx association for a series of γ- subunit chimeras. Chimeric subunits were constructed with the N-terminal portion of the γ subunit and the C-terminal portion from the subunit joined at the given junction point (see Experimental Procedures for a description of the chimeras). Each chimera was cotransfected with wild-type R, β, and δ subunits. kobs is the observed first-order association rate constant for [125I]-R-BgTx in the presence of the given concentration of waglerin-1, and kmax is the association rate constant in the absence of waglerin-1.
type , indicating that this intervening γ sequence does not contribute to Wtx-1 selectivity. The collective results indicate that determinants for the γ- affinity difference localize to three regions of the subunit: residues 1-65, 103-117, and 171-177.
7898 Biochemistry, Vol. 41, No. 25, 2002
Molles et al.
Table 1: Dissociation Constants for Wtx-1 Binding to Receptors with Chimeric γ- Subunitsa tested subunit
KD1 (nM)
γ γ74 65γ76 γ103 γ117 110γ114 γ165 γ171 γ177 b 56γ61b 57γ61b 58γ61b
9.8 36000 810 14.8 825 28200 134 11400 9300 37000 6.0 218 4.1 13.6
KD2 (µM) KD2/KD1
nH
∆KD1 ( wt)
20.4 22.7 12.7 31.7 26.8 10.9 11.9 6.0 5.9
1.2 1.2 1.1 1.0 1.7 -
3700 26 3700 7 82 44 3 1.5 2400 1 84 44 2 2900 1.3 2 14 1.3 2 1200 3.2 1 950 3.9 3 3800 1.0 2 4 36 1.1 3 0.7 0.6 1 2.3 0.5 2
2100 34 860 38 200 1820 55 1460 430
∆KD1 (γ wt)
n
a All experiments were performed on cells transfected with mouse wild-type R, β, and δ subunits and the given γ or subunit construct. KD1 is the high-affinity site dissociation constant, KD2 the lower-affinity site dissociation constant (when the two sites have the same affinity, they are reported as KD1), nH the Hill slope for constructs in which the nonlinear regression curve fit best to a one-site analysis, ∆KD1 ( wt) the fold difference in affinity between the KD1 for the tested subunit and the KD1 value for the wild-type subunit, ∆KD1 (γ wt) the fold difference in affinity between the KD1 with the tested subunit and the KD1 value with the wild-type γ subunit, and n the number of experiments. b Indicates experiments performed with Wtx-1 peptide amidated at C-terminus. Wtx-1-amide has ∼2-fold higher affinity at both sites than wild-type Wtx-1.
Previous work identified two regions containing determinants of ligand selectivity N-terminal to position 65. One region contains K34 in the γ subunit and the equivalent S36 in the δ subunit, which contribute to binding site selectivity for carbamylcholine (25) and R-conotoxin MI for fetal nAChR (13). However, because Lys is present at position 34 in both γ and subunits, it cannot contribute to Wtx-1 selectivity for these subunits. We therefore examined the second N-terminal region (15), between residues 55 and 60 of the subunit, using the 56γ61, 57γ61, and 58γ61 chimeras and the H61C point mutant. The 56γ61 chimera reduces affinity 40-fold compared to that of the wild-type subunit, implicating residues 57-61 in the subunit and corresponding residues in the γ subunit as sources of Wtx-1 selectivity (Table 1). To pinpoint selectivity determinants in the region of residues 56-61, we examined point mutations. The G57E point mutation reduced affinity 13-fold, and D59Q reduced affinity 5-fold compared to that of the wild-type subunit (Figure 3 and Table 2). Placing the two mutations in the same construct (G57E/D59Q) reduced affinity 76-fold, which is essentially the same as the 80-fold reduction found for the γ74 and γ103 chimeras, but greater than the 40fold reduction observed for the 56γ61 chimera. Thus, determinants in the N-terminal 74 amino acids are located at positions 57 and 59. If the protein scaffolds of the γ and subunits are superimposable, increases in affinity due to mutations in the subunit should be comparable in magnitude to the decreases in affinity for equivalent mutations in the γ subunit. The point mutations γE57G, γQ59D, and γE57G/Q59D increased affinity only 2-, 2-, and 5-fold, respectively, values not nearly as large as the reductions in affinity caused by the corresponding mutations in the subunit (Figure 3 and Table 2).
FIGURE 3: Waglerin competition with the initial rate of [125I]-Rbungarotoxin binding for a series of residues determining and γ subunit selectivity. All experiments were performed on HEK cells cotransfected with wild-type R, β, and δ subunits and the indicated mutated or γ subunit. Sequence analysis is depicted in detail in Figure 1. Arrows indicate the direction of shift in the binding curve resulting from the given mutation. Waglerin KD values for each receptor are given in Table 2. (A) to γ subunit point mutations. Residues in the subunit template were mutated at the selectivitydetermining residues to the corresponding amino acids found in the γ subunit. (B) γ to subunit point mutations. Residues in the γ subunit template were mutated at the selectivity-determining residues to the corresponding amino acids found in the subunit.
This difference between and γ subunits following mutation suggests the presence of additional selectivity determinants in the N-terminus of the subunit that either interact with residues at positions 57 and 59 or position Wtx-1 to interact with these determinants. To localize selectivity determinants within the second N-terminal region, residues 103-117, three -γ- chimeras were made: 110γ117, 110γ114, and 114γ117. Following cotransfection with complementary wild-type subunits, only the 110γ114 chimera was expressed well, and it reduced affinity 23-fold relative to that of the wild-type subunit (Table 1), indicating key determinants between positions 110 and 114. The residue at position 111 is a key selectivity determinant for R-conotoxin MI at the R-γ site in fetal AChR (13), and is Tyr in the subunit and Ser in the γ subunit. The point mutant Y111S reduced Wtx-1 affinity 4-fold, and Y115C, which replicates the position 115 difference between and γ subunits (Figure 1), reduced affinity 13-fold. The double mutation Y111S/Y115C reduced affinity 30-fold (Figure 3A and Table 2), close to the additive change in affinity for individual point mutants, and nearly identical to the difference in affinity between the γ103 and γ117 chimeras. Thus results from chimeras and
Waglerin-1 Binding to Nicotinic Receptors
Biochemistry, Vol. 41, No. 25, 2002 7899
Table 2: Dissociation Constants for the nAChR with γ or Residue Substitutions into or γ Subunits, Respectivelya γ subunit substitutions into an subunit template
KDR- (nM)
KDR-δ (µM)
KDR-/KDR-δ
nH
∆KDR-
n
G57E D59Q G57E/D59Q Y111S Y115C Y111S/Y115C D173F Y111S/D173F G57E/D173F G57E/D59Q/D173F G57E/D59Q/Y111S/Y115C/D173F
9.8 125 47 743 38 126 290 2710 8900 13800 17900 11500
20.4 18.0 33.9 34.8 23.2 14.7 27.4 23.9 -
2100 140 720 47 610 120 94 9 -
1.0 1.0 1.0 1.1
13 5 76 4 13 30 280 910 1400 1800 1200
26 3 3 2 2 3 3 3 1 3 2 2
subunit substitutions into a γ subunit template
KDR-γ (nM)
KDR-δ (µM)
KDR-γ/KDR-δ
nH
∆KDR-γ
n
γ γE57G γQ59D γE57G/Q59D γC61H γQ59D/C61H γS111Y γC115Y γS111Y/C115Y γE57G/C115Y γF172D γE57G/F172D γS111Y/F172D γE57G/Q59D/F172D γE57G/C115Y/F172D γE57G/Q59D/S111Y/C115Y γE57G/Q59D/S111Y/C115Y/F172D
36000 14700 11400 9200 27400 7700 20800 20700 26900 18200 7200 537 2510 525 407 15300 98.3
29.2 22.2 29.8 28.4 16.6
94 9 57 70 170
1.2 1.0 1.2 1.2 1.1 0.9 1.2 1.1 1.5 1.0 0.9 1.0 -
2.5 3 5 1.3 5 1.7 1.7 1.3 2.0 5 67 14 68 88 2 370
7 2 3 3 1 1 2 2 2 2 3 3 1 3 3 2 3
a All experiments performed on cells transfected with mouse wild-type R, β, and δ subunits and the given or γ subunit. KDR-, R- site KD; KDR-δ, R-δ site KD; ∆KDR-/γ, fold change in the mutant KDR-/γ value compared to the wild-type KDR- (∆KDR-δ values differed by less than 2-fold from wild-type KDR-δ); n, number of experiments. Results of experiments in which mutation of the or γ site yielded a KDR-/γ which superimposed on KDR-δ are listed in the KDR-/γ column only, though this value represents the composite Wtx-1 KD found at both the R-/γ and R-δ sites.
point mutants indicate residues at positions 111 and 115 are determinants of Wtx-1 selectivity. The γ117 chimera reduced Wtx-1 affinity to nearly that conferred by the wild-type γ subunit, and the four identified determinants, two major (Gly-57 and Tyr-115) and two minor (Asp-59 and Tyr-111), all lie within this N-terminal 117amino acid region. Although the quadruple mutant of these determinants was not made in the subunit, the sum of the respective affinity reductions for the four mutants fully accounts for the entire 3700-fold difference in affinity between R- and R-γ binding sites, as well as that between R- and R-γ117 sites. We therefore concluded that these four determinants account fully for Wtx-1 selectivity between R- and R-γ binding sites. However, as seen with the γE57G and γQ59D mutants, the γS111Y and γC115Y mutants did not significantly increase Wtx-1 affinity relative to that of the wild-type γ subunit (Table 2). Furthermore, Wtx-1 affinity conferred by the quadruple mutant, γE57G/ Q59D/S111Y/C115Y, did not change compared to that of the wild-type γ subunit (Table 2). Hence, within the N-terminal region containing residues 1-117, a large convergence of affinities between and γ can only be achieved by γ substitutions in the template, which reduces affinity, but not by substitutions in the γ template. Accordingly, we sought additional determinants of Wtx-1 affinity, and examined whether they affect contributions of the four identified selectivity determinants.
The γ165 and γ171 chimeras increased affinity 3-fold compared to those of γ117, γ177, or wild-type γ (Table 1), suggesting a minor determinant between positions 171 and 177. This region harbors a second determinant of R-conotoxin MI selectivity, γPhe-172/Asp-173/δIle-178 (13). Paradoxically, the D173F point mutant, far outside the N-terminal 117-amino acid region thought to harbor most of the selectivity determinants, diminished affinity by 280fold. This unexpected reduction in affinity is significantly greater than for any individual point mutation studied thus far, far greater than the 4-fold affinity change between the γ171 and γ177 chimeras. Analogously, the 5-fold increase in affinity observed for the homologous residue replacement in the γ subunit, γF172D (Figure 3B and Table 2), while consistent with the small affinity difference between the γ173 and γ177 chimeras, was far smaller than the 276fold decrease in affinity for the converse residue replacement in the subunit, D173F. The γ- chimeras and single point mutations reveal three regions of primary sequence that determine the high affinity of waglerin-1 for the R- versus the R-γ site: (a) N-terminus to position 74, (b) between positions 103 and 117, and (c) between positions 171 and 177. When the subunit is mutated at positions 57, 59, 111, 115, and 173 to the corresponding residues in γ, the individual reductions in affinity fully account for the corresponding reductions suggested by the chimeras. When these five mutations were
7900 Biochemistry, Vol. 41, No. 25, 2002
Molles et al.
Table 3: Waglerin-1 Dissociation Constants for δ Subunit Substitutions into an Subunit and with -δ Chimerasa δ subunit substitutions into an subunit template
KDR- (nM)
KDR-δ (µM)
KDR-δ/ KDR-
∆KDR-
n
K34S G57D D59A G57D/D59A D173I K34S/D173I G57D/D173I G57D/D59A/D173I K34S/G57D/D59A/D173I
9.8 55.3 288 10.8 77.0 1040 360 625 1200 1970
20.4 33.0 26.9 18.1 22.4 22.9 25.3 29.3 42.5 61.7
2100 600 93 17000 290 22 70 47 35 31
6 29 1 8 106 37 64 120 200
26 2 3 2 2 3 3 3 2 3
-δ chimeras and substitutions KDR- KDR-δ KDR-δ/ (nM) (µM) KDR- ∆KDR-δ n into a δ subunit template δI178D 117δ δD114E/I178D δS115G/I178D δT119S/I178D δD114E/S115G/T119S δD114E/S115G/T119S/I178D 117δI178D 65δ 65δI178D δ3165δI178D
18.7 16.6 23.4 14.0 18.5 7.1 4.8 12.1 32.3 23.2 5.8
3.0 4.0 2.2 2.3 1.2 5.9 2.5 0.12 6.9 0.13 0.24
160 240 95 160 67 830 760 10 210 6 41
7 5 9 9 17 4 8 170 3 160 84
7 3 2 2 3 2 2 2 5 5 2
a The top section lists results of experiments performed on cells transfected with wild-type R, β, and δ subunits and the given subunit. The bottom section lists results of experiments performed on cells transfected with wild-type R, β, and subunits and the given δ subunit chimera and/or substitution. Column labels are as defined in Table 2.
incorporated into a single subunit, the affinity decreased to within 3-fold of that of the wild-type γ subunit (Figure 3A and Table 2). Conversely, whereas mutation of these same residues indiVidually in the γ subunit fell far short of the high affinity expected for the wild-type R- site, the corresponding quintuple mutant in the γ subunit, γE57G/ Q59D/S111Y/C115Y/F172D, increased Wtx-1 affinity to within 10-fold of that of the wild-type R- site (Table 2 and Figure 3B). Thus, the collective results delineate multiple determinants of Wtx-1 selectivity in and γ subunits and, moreover, show that their contributions are interdependent. This interdependence of residues is analyzed in the Discussion. δ- Subunit Determinants for Waglerin-1. Wtx-1 selects between R- and R-δ sites in the adult nAChR by 2100fold because of different contributions of and δ subunits. Although the δ and subunits exhibit less residue identity than the γ and subunits and occupy different positions in the ordering of the subunits around the pentamer, we tested the hypothesis that residues at equivalent positions of these subunits govern Wtx-1 selectivity for sites in the adult nAChR. We constructed chimeras and point mutants based on the δ subunit, cotransfected them with wild-type R, β, and subunits, and assessed Wtx-1 binding. To test the contribution of the N-terminal region, we examined the 65δ chimera, but found only a 3-fold increase in affinity compared to that of wild-type δ. Point mutations of the identified γ/ determinants, δD59G and δA61D, failed to increase affinity. The converse mutation in the subunit, G57D, decreased affinity 29-fold, whereas the D59A mutant was without effect (Table 3). Thus, as observed for mutations in γ and subunits, mutation of residue 59 in the
δ subunit had a markedly different consequence than mutation of the equivalent residue in the subunit, again suggesting multiple residues interact to confer Wtx-1 selectivity. The D173F mutation, which substitutes the residue at the equivalent position in the γ subunit, reduces affinity by 280fold, the largest reduction for any single-residue change (Table 2). The corresponding to δ mutation, D173I, reduces affinity by 106-fold, but the converse δ to mutation, δI178D, increases affinity only 7-fold (Table 3). These results provide additional evidence that multiple residues interact to confer Wtx-1 selectivity for sites in the adult nAChR. Thus, subsequent mutations were made in a construct containing the δI178D mutation to investigate cooperative interactions. We initially combined the 65δ chimera with the δI178D point mutant to give 65δ/I178D. This construct increased affinity 156-fold compared to that of wild-type δ (Table 3), which is far greater than the 21-fold increase expected from additive contributions (3-fold for 65δ and 7-fold for δI178D). Hence, one or more residues N-terminal to position 65 appear to be energetically coupled to δI178. Additional constructs, 117δ and 117δ/I178D, gave affinities essentially identical to those of the 65δ and 65δ/I178D constructs, respectively (Table 3), suggesting that no additional /δ selectivity determinants are present between positions 67 and 119 of the δ subunit. To test further for interacting residues, we started with the mutation δI178D and introduced a series of δ to subunit mutations at the following single sites: δD59G, δA61D (Table 5), δH60I, δV63H, and δS65Y. No evidence for interacting residues was obtained for any of these double mutants, although the D59G/I178D mutation increased affinity 3-fold compared to that of the δI178D mutation alone. The triple mutation δD57G/A61D/I178D increased affinity 46-fold compared to that of wild-type δ (Table 5), the largest increase of any combination of point mutants in the δ subunit. Only one other determinant of ligand selectivity N-terminal to position 55 has been described: Lys-34 in γ and subunits and the equivalent Ser-36 in the δ subunit (13, 25). The point mutant K34S reduced affinity for Wtx-1 by almost 6-fold (Table 3), but the converse mutant δS36K mutant paradoxically reduced affinity by 2-fold. By comparison, the δS36K/ I178D double mutant enhanced affinity 4-, 60-, and 30-fold over those of the δI178D mutant, the δS36K mutant, and the wild-type δ subunit, respectively. That δS36K enhances rather than reduces affinity when combined with δI178D indicates that these two oppositely charged residues interact in contributing to Wtx-1 selectivity. To look further for /δ selectivity determinants, we examined the three residues that differ between positions 113 and 119 of the δ subunit, as two residues in this region contribute to the /γ selectivity difference for Wtx-1. We mutated δD114, δS115, and δT119 to the corresponding residues in the subunit and combined each with the δI178D mutant. For all three double mutations, no change in affinity was observed compared to that of δI178D alone (Table 3). The triple mutant, δD114E/S115G/T119S, increased affinity 3-fold, but this increase was not maintained in the δD114E/
Waglerin-1 Binding to Nicotinic Receptors
Biochemistry, Vol. 41, No. 25, 2002 7901
Table 4: Pairwise Interaction Energies (Linkages) for Multiple Mutations in the and γ Subunit Templatesa subunit template
mut1
to δ mutations G57D/D59A K34S/D173I G57D/D173I G57D/D59A/D173I G57D/D59A/D173I K34S/G57D/D59A/D173I K34S/G57D/D59A/D173I to γ mutations G57E/D59Q Y111S/Y115C G57E/D173F G57E/D59Q/D173F G57E/D59Q/D173F G57E/D59Q/ Y111S/Y115C/D173F
mut2
Ω
∆∆GINT (kcal/mol)
G57D K34S G57D G57D/D59A G57D/D173I G57D/D59A/D173I G57D/D59A
D59A D173I D173I D173I D59A K34S K34S/D173I
4.1 16.3 48.8 6.8 1.7 3.4 1.4
-0.84 -1.65 -2.29 -1.13 0.33 -0.73 -0.21
G57E Y111S G57E G57E/D173F G57E/D59Q G57E/D59Q/D173F
D59Q Y115C D173F D59Q D173F Y111S/Y115C
1.2 1.7 2.5 3.7 11.5 46.1
0.13 -0.30 -0.54 -0.77 -1.44 -2.26
γ subunit template
mut1
mut2
Ω
∆∆GINT (kcal/mol)
γ to mutations E57G/Q59D S111Y/C115Y E57G/C115Y E57G/F172D E57G/Q59D/F172D E57G/Q59D/F172D E57G/C115Y/F172D E57G/C115Y/F172D E57G/Q59D/C115Y E57G/Q59D/S111Y/C115Y/F172D E57G/Q59D/S111Y/C115Y/F172D
E57G S111Y E57G E57G E57G/Q59D E57G/F172D E57G/F172D E57G/C115Y E57G/Q59D E57G/Q59D/F172D E57G/Q59D/S111Y/C115Y
Q59D C115Y C115Y F172D F172D Q59D C115Y F172D C115Y S111Y/C115Y F172D
2.2 2.2 2.2 5.5 3.5 3.4 1.3 9.0 2.2 4.0 31.2
0.46 0.48 0.45 -1.00 -0.74 0.72 0.16 -1.29 0.47 -0.82 -2.03
a All experiments were performed on cells transfected with mouse wild-type R, β, and δ subunits and the given (top section) or γ (bottom section) subunit mutation(s). Actual KD values are given in Tables 2 and 3. mut1 and mut2 are the mutation(s) represented in Scheme 1 of the text. Ω is defined in eq 2 of the text, and its reciprocal is shown for values
